Matrix for receiving a tissue sample and use thereof

ABSTRACT

A custom-made matrix suitable for receiving a tissue sample is described, as well as the use thereof to obtain a multiplex histological preparation. The disclosure also relates to a multiplex biopsy array comprising tissue and/or cell samples arranged in a matrix material and to a method for the preparation of a multiplex biopsy array. Methods for preparing blocks of matrix material to be used in multiplex biopsy arrays are also described, as well as methods for loading biopsy samples in the blocks, and methods for treating and processing the blocks to form biopsy arrays. The biopsy arrays made using the block of matrix material can be used to prepare sections and slides for histological procedures, including quantitative analyzes and parallel processing.

CLAIM OF PRIORITY

This application claims benefit or priority to U.S. provisional PatentApplication Ser. No. 61/502,513, filed Jun. 29, 2011, titled “MATRIX ANDMETHOD FOR THE PREPARATION OF MULTIPLEX BIOPSY ARRAYS,” the disclosureof which is hereby incorporated herein in its entirety by thisreference.

CROSS-REFERENCES

Not Applicable.

TECHNICAL FIELD

The disclosure relates to a custom-made matrix suitable for receiving atissue sample and to the use thereof to obtain a multiplex histologicalpreparation. The disclosure also relates to methods for preparing blocksof matrix material to be used in constructing multiplex biopsy arrays,methods for loading tissue samples in the blocks, and methods fortreating and processing the blocks and arrays in preparation foranalysis.

BACKGROUND

The tissue sample may result from a needle biopsy (sometimes referred toas tru-cut biopsy), fine needle aspirate or an explant. A needle biopsyis a common medical test involving the removal, with the aid of a hollowneedle, of a representative sample of cells or tissues from a livingsubject for examination to determine the presence or extent of adisease. Fine needle aspiration (FNA) is a diagnostic proceduresometimes used to investigate superficial (just under the skin) lumps ormasses or hollow organs. According to this technique, a thin hollowneedle is inserted into the mass for sampling of cells that, after beingstained, will be examined under a microscope. A biological sampleharvested from a piece or pieces of tissue is called an “explant.” Theexplant and/or biopsy sample is subjected to treatments, such asfixation, sectioning and staining, and then examined by a pathologist.The tissue sample can also be analyzed chemically. For the sake ofbrevity, the term “biopsy” will be used throughout this application withthe understanding that it refers to tru-cut biopsies as well as to fineneedle aspirates and free-form explants.

Patterned biopsies play a major role in the early detection of varioustypes of cancer (i.e., prostate, breast, thyroid, skin, intestine, lung,stomach, etc.). Maintaining site-specific information regardingindividual biopsy cores is of critical importance. While individualprocessing of biopsies by conventional methods is prohibitivelyexpensive, current methods of parallel processing (tissue microarrays,color coding, multi-compartment cassettes, etc.) are either notaccurate, cumbersome, or both.

A standard biopsy method uses a needle to remove a biopsy sample forexamination. A tru-cut needle biopsy removes small but solid samples oftissue using a hollow “core” needle. In a core biopsy, a smallcylindrical sample of tissue is removed preserving the histologicalarchitecture of the tissue's cells. This is important when dealing withbiopsies (e.g., prostatic, breast) where one has to report thepercentage of malignant tissue. The samples are small, and fragile, andtend to curl during processing, making preparation of the sample forexamination difficult.

In the United States, there are approximately 1 million patientsrequiring biopsies for early diagnosis of prostate cancer. On average,10 to 21 biopsy samples are taken per patient with suspicion of prostatecancer and their precise location has to be recorded. In some cases“prostate mapping”/saturation biopsies are performed (up to 60 to 80biopsy samples per patient). Utilizing the standard biopsy technique (inwhich one, or maximum two, biopsies are processed at a time) results ina huge number of paraffin blocks, requiring a large number of sectionsand slides, high expenditures in terms of consumables, manpower forprocessing and time spent by the pathologists for interpreting theslides. Moreover, in order to provide the required 3-D reconstructionanalysis of malignancy, it is very important to record and map thelocation of the sections relative to the original biopsy sample.

Traditionally, when dealing with biopsy samples, the recommendedapproach is to process the samples by embedding individually in asupporting material such as a paraffin block. The paraffin blockscontaining the individual samples are then sectioned with a microtome toproduce thin sections that will be placed on a microscope slide, stainedas needed and examined under a microscope. When sectioning the paraffinblocks, one runs the risk of not intercepting the tissue sample and/orlosing too much of the sample before a (quasi) complete section isproduced. For example, when dealing with prostate biopsies, currentrecommendations require three different sections (“step” sections, or“levels”) taken at approximately 50 microns apart. Usually, two to threeadditional sections (unstained) are saved at every step/level forfurther staining, if required by the pathologist. Sufficient materialshould remain in the block for further study as well as for archiving,litigation, etc. Because of the scarcity of the material in the sample,only adhesive-coated slides (i.e., 10 to 20 times more expensive thanregular variety) are employed, to minimize the risk of accidentallylosing the sections during staining.

In recent years, trans-rectal ultrasound-guided (TRUS) systematic needlebiopsy has emerged as a new gold standard in prostate cancer diagnosis,to such an extent that statistical performance values (sensitivity,specificity, positive and negative predictive values) of all otherdiagnostic tests, like digital rectal examination (DRE) orprostate-specific antigen (PSA) are computed according to the outcome ofbiopsy examination. A computerized model of the prostate, includingmapped sections from 159 whole-mount radical prostatectomy specimens,subjected to systematic histopathology examination showed that thesix-core biopsy technique fails to identify 26.8% of the tumors. Even onrepeated examination, this method resulted in failure of tumoridentification in 27 of 118 prostate cancer patients, equivalent to 23%of total cases demonstrated following radical prostatectomy. From aplethora of recent studies comparing results of different biopsystrategies regarding the number and location of cores, the emergingconsensus, based on clinical trials, ex vivo biopsy approaches, as wellas various computer simulations and mathematical models, is to take atleast ten biopsy cores, focusing the biopsies laterally at the base,mid-gland, and apex of the prostate, with mid-lobar biopsy cores at thebase and apex, and adjusting the number of cores taken according toprostate volume and age of the patient. Some investigators advocate evenmore aggressive biopsy schemes, with more than 12 cores, up to asaturation biopsy (>20 cores), especially on repeat biopsies, reportingeven higher cancer detection rates. However, despite the obvious needfor multiple biopsy cores per patient, due to high expenses and limitedresources, the number of biopsies taken and sections prepared perpatient is typically reduced to the minimum required for an acceptableprecision of the diagnosis and/or the maximum resources available withinan institution. Thus, a system for rapid, cost-effective preparation andanalysis of multiple cores at multiple levels is needed.

An array is an organized fashion of multiple tissue and/or cell samplesthat can be used in various histological techniques includingtopographical staining, (immuno)histochemistry, immuno-fluorescence, andin situ hybridization. Tissue and cell arrays are powerful tools becausethey allow simultaneous screening of numerous tissue or cell samples.The value of this type of technology is that testing can be done on manysamples in a timely manner with consistency. This allows forhigh-throughput histological screening or analysis.

There are a number of methods that describe the construction of tissueor cell arrays including: the “sausage” method (see Battifora, “Themultitumor (sausage) tissue block: novel method for immunohistochemicalantibody testing” (1986) Lab. Invest. 55:244; and U.S. Pat. No.4,820,504); paraffin-to-paraffin transfer methods (see Kononen et al.,“Tissue microarrays for high throughput molecular profiling of tumorspecimens” (1998) Nature Medicine 4(7):844-847); the “honeycomb” method(see K. Petrosyan and M. F. Press, “Multispecimen tissue blocks inpathology: an improved technique of preparation” (1997) Lab. Invest.77(5):541-542); and the use of liver as a recipient matrix for the array(see Musat-Marcu et al., “Inhibition of apoptosis afterischemia-reperfusion in rat myocardium by cycloheximide” (1999) J. Mol.Cell. Cardiol. 31:1073-1082). Also see U.S. Pat. No. 4,647,543; Millerand Groothius, A.J.C.P. 96:228-232; Sundbland, A.J.C.P 102:192-193;Patent Application No. WO1999IUS9912537; Patent Application No.PCT/US99/04000; Patent Application No. WO1999/WO0004001 and U.S. patentapplication Ser. No. 1987000110818.

The “sausage” technique involves combining multiple tissue samples froma deparaffinized block into a single composite “sausage” held togetherwith a wrapper of intestinal casing. The multiple tissue sausages arere-paraffinized, sectioned and then mounted on slides. This procedureallows hundreds of tissue samples to be tested simultaneously. Eventhough this approach is valuable, it has a number of inherentdisadvantages. For instance, the technical effort and time required toprepare the composite sausage causes difficulties. In addition, the needto de-paraffinize and re-paraffinize the tissue samples could lead to aloss of antigens. There are problems in maintaining the spatialrelationships among the different tissue samples and working with smallspecimens such as cells, because of the flexible nature of theintestinal casing.

There have been a number of other paraffin-to-paraffin ordouble-embedding techniques that have evolved to fix some of theproblems with the “sausage” technique (see U.S. Pat. Nos. 4,914,022 and5,002,377). A common multi-specimen technique involves the preparationof standard paraffin blocks of tissue specimens where core samples arethen removed from these blocks and re-embedded into a recipient paraffinblock to create the tissue array (see U.S. Pat. No. 4,914,022). Thistechnique can be used to test multiple tissue samples from multiplesources at the same time. In addition, it is possible to take a tissuechip, which is a thin section of the tissue array, and parallel processa number of samples at the same time with a variety of stains ormolecular markers. The problem with this technique is that in order toprepare the tissue chips, custom-built equipment is required, whichincludes a computer-controlled micro-stage. In addition, the recipientparaffin block cannot be cut unless the adhesive tape technique isemployed. The adhesive tape technique is expensive and time-consuming,and because the cut sections require special treatments before staining,there is a risk of compromising the accuracy of many applications.Another problem with paraffin-to-paraffin techniques is that therecipient paraffin block cannot cut efficiently and, thus, a number ofsections are lost. Also, serial sectioning of the recipient block toproduce ribbons is virtually impossible. Another limitation of thistechnique is that the tissue samples must be paraffinized and thenre-paraffinized, thus, it is not possible to use fresh samples to createthe microarray and antigens might be lost in the process. This techniquecan also be used to construct cell arrays; however, the samedisadvantages exist as found when constructing tissue arrays (see M.Cottler-Fox and C. H. Fox, J. Infect. Dis. (1991), 164:1239-1240).

The “honeycomb” technique to create tissue arrays has the advantage ofbeing able to employ fresh or fixed tissue without prior embedding. Thistechnique uses a multi-chambered mold (“honeycomb”) made with plastowax.Small tissue specimens are placed in the equal-sized spaces of the mold,and then the molds are embedded with PARAPLAST®. The multi-specimentissue blocks are processed, sectioned and stained using conventionalmethods. The problem with this technique is that precise orientation ofthe individual samples is not possible. As a result, its applicationsare very limited and, as such, it is not widely used. Like all previousmethods, it requires infiltration followed by embedding, for example, inparaffin.

Liver tissue has previously been proposed as a support for facilitatingsectioning of tissue samples (see, e.g., Manfred Gabe, “HistologicalTechniques,” p. 125 (Springer-Verlag 1976), and Musat-Marcu et al.,“Inhibition of apoptosis after ischemia-reperfusion in rat myocardium bycycloheximide,” J. Mol. Cell. Cardiol. 31:1073-1082 (1999). Tissue andcell arrays can be constructed using liver as the recipient matrix. Thistechnique works for fixed, fresh, or paraffinized tissue or cellsamples. The problems with this technique include the low efficiency ingenerating the required matrices and the occasional hidden “defects” inthe liver matrix, such as biliary ducts, blood vessels and collagensepta that results in a loss of samples. In addition, there is possiblecross-reactivity between the matrix and biopsy samples.

Thus, although tissue arrays and cell arrays are powerful tools to allowsimultaneous screening of numerous tissue or cell samples there are nosimple methods for creating arrays using core biopsy sample or free-formexplants. Thus, there is, therefore, a need for improved matrixmaterials and techniques for creating biopsy arrays of fixed, fresh, orparaffinized samples where such techniques and materials areinexpensive, reliable, consistent and simple and avoid the limitationsand complications of the prior techniques.

BRIEF SUMMARY

Embodiments of the present disclosure provide an inexpensive and precisemethod for parallel processing of prostate core biopsies that generateshigh yields and minimal losses while maintaining site-specificinformation regarding biopsy cores.

In one aspect, the present disclosure relates to a custom-made matrixsuitable for receiving a tissue sample and to the use thereof to obtaina multiplex histological preparation. The disclosure also relates tomethods for preparing blocks of matrix material for preparing multiplexbiopsy arrays, methods for loading biopsy samples in the blocks, andmethods for treating and processing the blocks and arrays in preparationfor analysis.

In another aspect, the present disclosure provides a tissue matrixemployed as a recipient matrix for biological samples, such as tissueand/or cell sample(s). In one embodiment, the disclosure provides amatrix that can be used for receiving at least one biopsy sample,wherein the biopsy sample is of a different origin from the tissuematrix. In addition, the disclosure provides a tissue matrix that can beused to construct a biopsy array.

In another embodiment, the disclosure also concerns a method forpreparing a biopsy sample and a recipient matrix for use in thedisclosure, and to a method for constructing a biopsy array using thematrix. The disclosure includes the use of the biopsy array or embeddedbiological sample, for instance, in histological procedures. In oneaspect, the disclosure provides a histological preparation comprising arecipient matrix of the disclosure and a tissue and/or cell sample. In afurther embodiment, the recipient matrix can be used in the productionof single or multi-specimen/multi-use preparations. In a preferredembodiment, the recipient matrix is comprised of processed animaltissue. For the purposes of this specification, animal tissue is meantto comprise, inter alia, porcine tissue, avian tissue, fish tissue, etc.

In another embodiment, the disclosure relates to a processed animaltissue matrix that can be used to embed a biological sample or constructa tissue and/or cell array. The processed animal tissue used in thematrix consists of a suitable blend of proteins, fat and water toproduce a matrix with minimal shrinking and good cutting properties.

In another embodiment, the disclosure relates to the use of SPAM®processed pork to construct the processed animal tissue matrix. Thelabeled ingredients in the classic variety of SPAM® are chopped porkshoulder meat, with ham meat added, salt, water, modified potato starchas a binder, and sodium nitrite as a preservative. An advantage of SPAM®processed meat is that it allows the matrix and tissue samples to bereadily distinguished when biopsy samples are embedded therein. Inaddition, the size and shape of the matrix can be varied and, thus,allows flexibility in preparing the biopsy array.

In another embodiment, the disclosure provides a method for preparingthe processed animal tissue for use in creating biopsy arrays. Theprocessed animal tissue can be treated using known histologicaltechniques. The processed animal tissue can be fresh, fixed or embedded.Receptacles are formed in the processed animal tissue matrix, and tissuebiopsies or cells are placed therein, with or without the aid of avacuum, to create the matrix.

In another embodiment, the disclosure provides a method of treating andobtaining a biological sample, such as a biopsy sample to be used in thematrix to create the array. An advantage of this disclosure is thatsmall samples are as easily handled as larger samples. Thus, it ispossible to use tissue samples or cell samples in the array. Inaddition, the biopsy samples can be in a variety of preparative forms,including fresh, fixed or paraffin-embedded, and, thus, the problems ofprior methods that require each tissue sample to be paraffinized andre-paraffinized are avoided. A wide assortment of sources can be used assamples, such as normal organs/tissues, tumors, blood, bodily fluids orsecretions, primary cell cultures and cell lines.

In another embodiment, the disclosure provides biopsy arrays made usingthe processed animal tissue matrix. The biopsy arrays that areconstructed using the processed animal tissue matrix are flexible intheir design. The array may be composed of tissues or cells or acombination of both tissues and cells. The size and shape of the arraycan be varied to support different sizes and shapes of biopsy samples.The arrays can be custom designed to compare different samples from asingle species, the same cell or tissue type from different species, orthe same cell or tissue type from the same species at differentdevelopmental times. Advantages of this disclosure include ease ofsectioning the matrix and minimal shrinkage.

In another embodiment, the disclosure provides a method for preparingthe biopsy arrays. The processed animal tissue matrix is preferably in asimilar or subsequent preparative stage as the tissue and/or cell samplebefore the sample is arranged in the processed animal tissue matrix tocreate the biopsy array. In one embodiment, the cell or tissue isarranged in the receptacle in the processed animal tissue matrix with orwithout the aid of a low vacuum. A thin layer of additional processedanimal tissue matrix, which is preferably in a similar preparative stageas the original processed animal tissue matrix, can be used as a lid tocover the receptacle when needed. The biopsy array can then be processedusing known histological techniques. The present method creates a biopsyarray that can be easily cut to create consistent duplicate samples orarrays for analysis or other purposes. Therefore, unlike other methods,it can be used efficiently for parallel analysis or quantitativeanalysis of various biopsy samples. This new method overcomes thedisadvantages noted above in the construction and use of biopsy arrays.This method is inexpensive and avoids the problems of other methods.

In another embodiment, the disclosure relates to a matrix material forpreparing multiplex biopsy arrays comprising the matrix material. Thedisclosure also relates to methods for preparing blocks of matrixmaterial for preparing multiplex biopsy arrays, methods for loadingbiopsy samples in the blocks, and methods for treating and processingthe blocks and arrays in preparation for analysis.

In another embodiment, the disclosure includes a histologicalpreparation comprising a matrix suitable for receiving a biopsy samplein combination with at least one biopsy sample arranged in the matrix,wherein the biopsy sample is of a different origin from the tissuematrix.

In another embodiment, the disclosure provides a method for preparing ahistological preparation comprising a matrix material suitable forreceiving a biopsy sample in combination with at least one biopsy samplearranged in the matrix, wherein the biopsy sample is of a differentorigin from the matrix material. The method includes the followingpreparative steps: fixation, dehydration, infiltration with atransitional solvent, and embedding in a binding agent when the biopsyarranged therein is in a similar or subsequent preparative state as thematrix material.

In another embodiment, the disclosure provides a method for preparing ahistological preparation comprising a matrix material suitable forreceiving a biopsy sample in combination with at least one biopsy samplearranged in the matrix, wherein the biopsy sample is of a differentorigin from the matrix material. The method includes preparing a frozensample without fixation, dehydration, clarification or embedding.

In another embodiment, the disclosure provides methods for use of thebiopsy arrays. The biopsy arrays allow for simultaneous histologicaltesting of multiple individual biopsy samples on a single slide. Uses ofthe disclosure include creating sections and slides of a variety ofbiopsy samples. These sections or slices can be used with chemical orbiological stains, histochemistry, genetic probes, and other reagents.

In one embodiment, the biopsy array is paraffin-embedded. The processedanimal tissue array is flipped over in a base mold, and the paraffinblock is prepared using known procedures. The block can be cut and usedaccording to known histological techniques. In one embodiment, the blockis cut into sections. The technique of sectioning is known to personsskilled in the art.

In another embodiment, the disclosure provides a use of the matrix tocreate biopsy arrays that, in turn, can be used for histologicalpurposes. Histological purposes are known to persons skilled in the art.Such purposes include immunochemistry, immuno-fluorescence, in situhybridization, fluorescence in situ hybridization and the use of geneticprobes, chemical and biological reagents and stains using techniquesknown to those skilled in the art. In addition, the disclosure can beused to create slides, sections and tissue banking systems, which can beused in histological and quantitative analyses.

In one embodiment, a tissue biopsy array created using the processedanimal tissue matrix has less variation in results because the samplesare treated simultaneously. The present disclosure has a number ofadvantages including greatly enhanced efficiency and speed for tissuetesting; greatly decreased cost for multiple tissue testing; greateconomies in the use of tissue samples, reagents and testing materials;great flexibility and ease of constructing biopsy arrays; lack of needfor deparaffinization and re-embedding; ability to use tissues or cellsas sources of samples for the array; and the ability to use fresh, fixedor paraffinized biopsy sources. In one embodiment, it also enables oneto obtain serial sections (ribbons), for instance, as thin as 2 μm. Inanother embodiment, a biopsy array can be produced in less than 8 hours,more preferably between 8 and 24 hours. When using banked tissue(transitional solvent), an arrayed block can be generated within 3 hoursfor paraffin embedding, and in 10 to 15 minutes for frozen preparations.

This disclosure includes a matrix material that can be used to reliablyand inexpensively create biopsy arrays. The disclosure also relates tothe methods of constructing the biopsy arrays using the matrix materialand the use of the arrays in histological procedures. In a preferredembodiment, the matrix material is comprised of processed animal tissue.In one embodiment, the disclosure provides the use of processed animaltissue as a recipient matrix of a tissue and/or cell sample forhistological purposes.

Thus, the present disclosure provides a new matrix material and methodsfor preparing multiple biopsy arrays. These and other objects, featuresand advantages of the disclosure will be apparent from the drawings anddetailed description that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a perspective view of a matrix block for receiving biopsysamples according to an embodiment of the disclosure.

FIG. 1B illustrates the loading of biopsy samples into the matrix blockof FIG. 1A.

FIG. 1C illustrates the application of a sponge to the matrix block ofFIG. 1A.

FIG. 1D illustrates the matrix block of FIG. 1A and sponge of FIG. 1C incombination with a tissue cassette.

FIG. 1E illustrates a slide prepared from a section of the matrix blockand biopsy samples of FIG. 1A according to an embodiment of thedisclosure.

FIG. 1F is a picture of a sample slide prepared utilizing the matrixblock of this disclosure.

FIG. 2A is a perspective view of an alternative matrix block accordingto an embodiment of the disclosure.

FIG. 2B is a perspective view of an alternative matrix block accordingto an embodiment of the disclosure.

FIG. 2C is a perspective view of an alternative matrix block accordingto an embodiment of the disclosure.

FIG. 2D is a perspective view of an alternative matrix block accordingto an embodiment of the disclosure.

FIG. 3A is a perspective view of an alternative matrix block accordingto an embodiment of the disclosure.

FIG. 3B is a schematic view of a method and apparatus for loading biopsysamples into the matrix block of FIG. 3A according to an embodiment ofthe disclosure.

FIG. 3C illustrates the matrix block of FIG. 3A in combination with atissue cassette and sponge.

FIG. 3D is a perspective view of a variation of the alternative matrixblock of FIG. 3A according to an embodiment of the disclosure.

FIG. 4A is a perspective view of an alternative matrix block accordingto an embodiment of the disclosure.

FIG. 4B is a perspective view of an alternative matrix block accordingto an embodiment of the disclosure.

FIG. 4C shows a plurality of biopsy cores loaded in a matrix block.

FIG. 4D shows a grid array of biopsy cores prepared for histologicalanalysis.

FIG. 4E shows a grad array of biopsy cores prepared for histologicalanalysis.

DETAILED DESCRIPTION

This disclosure refers to a histological preparation comprising a matrixmaterial suitable for receiving a tissue and/or cell sample to thematrix material itself and to a tissue/cell array comprising at leastone tissue and/or cell sample arranged in the matrix. In one embodiment,the tissue and/or cell sample is of a different origin from the matrixmaterial.

A histological preparation is known to persons skilled in the art. Itincludes blocks, sections and slides of various tissue and/or cellsamples prepared using histological techniques or practices. There aremany histological techniques known to persons skilled in the art. In oneembodiment, the following histological technique is used to create ahistological preparation of a biopsy sample. A biopsy sample is obtainedand then prepared using the ensuing preparative stages preferably inthis order: fixation, dehydration, if required, infiltration with atransitional solvent, and embedding in a binding agent. The tissue canthen be sectioned as desired. A person skilled in the art wouldunderstand that variations of such method and other histologicalpreparation techniques suitable for use in the disclosure are available.Further, such a person would understand that depending on the tissue,cell or use thereof, certain steps may be added or omitted. One of theadvantages of this disclosure over the prior art is that the sametechnology could be used for frozen preparations, where no fixation,dehydration, clarification or embedding is employed.

“Fixation” as used herein refers to treating the sample with a fixative.Fixatives are well known to persons skilled in the art. Fixativesinclude physical agents such as freezing in liquid nitrogen,dehydration, or microwaving, or chemical agents such as acids (e.g.,picric, nitric, acetic), alcohols (e.g., ethanol), aldehydes (e.g.,formaldehyde, glutaraldehyde) and others (e.g., mercuric chloride) or acombination thereof (e.g., formaldehyde/ethanol mixtures). Fixativesusually operate by denaturing, coagulating or cross-linking the proteinsfound in the biological samples. Fixatives may work by stopping all liveprocesses. They may prevent decay, maintain the spatial distribution ofconstituents or allow and/or facilitate the eventual staining of theresulting sections. Persons skilled in the art would be familiar withsuitable fixatives for use in this disclosure.

Dehydration is a process known to persons skilled in the art. One methodof dehydration is infiltrating the sample with graded concentrations ofa dehydrating agent such as ethanol, such as at 50%, then 70%, then 90%and finally 100%/ethanol. A person skilled in the art would know thatmany other dehydrating agents and methods of dehydration would besuitable for this disclosure.

Transitional solvents are known to persons skilled in the art. They arecommonly used after the dehydration step but before the embedding step.A transitional solvent is selected on the basis of its miscibility withboth the dehydrating agent (e.g., ethanol) and the binding agent (e.g.,paraffin). Such an agent may not be necessary if the dehydrating agentis miscible with the binding agent. In a common technique, the sample isinfiltrated with a transitional solvent after dehydration. In apreferred embodiment, transitional solvents, such as hydrocarbonsolvents that are miscible with both ethanol and paraffin, are used. Thetransitional solvent is then commonly removed and replaced by theembedding agent (e.g., molten paraffin) from the sample by heating thesample with or without the aid of a vacuum.

Embedding materials include paraffin, cellulose, nitrate, gelatin,agarose, epoxy resins, carbowax, and soaps. One purpose of bindingagents is to confer strength and support to the arrays duringsectioning. Paraffin and other waxes and resins have the additionalbenefit of preventing water vapors from deteriorating the constituentsof the samples. “Binding agent” as used herein includes embeddingmaterials.

Sectioning is a technique known to persons skilled in the art and itincludes cutting the sample into slices. In one embodiment, the slicescan be as thin as 2 μm.

The preparative stages that have been given as examples are merelysamples of possible histological techniques that can be used to createhistological preparations. A person skilled in the art would know thatvariations of these techniques exist and that various tissue and/or cellsamples can be prepared using alternative techniques and methods knownto persons skilled in the art. A person skilled in the art would alsoappreciate that different matrices and/or biopsy samples or uses thereofmay necessitate the use of different preparative methods, solvents, andagents.

“Different origin” refers to the use of a type of cell and/or tissuesample to be arranged in the matrix that is different from the type oftissue comprising the matrix. Preferably, the matrix is of an originthat does not cross react with the biopsy sample(s). For instance, thetissue and/or cell samples may be of animal origin, while the matrix iscomprised of processed animal tissue, or the tissue and/or cell samplesmay be from one species, while the matrix is comprised of tissue fromanother species.

“Array” as used herein refers to tissue and/or cell samples arranged ina matrix material. In one embodiment, the purpose of the array is toarrange the samples in a defined spatial orientation so that they can beidentified unambiguously. In another embodiment, the purpose is to embedcell samples and to prevent deformation of cell or tissue samples duringprocessing. In addition, the array can be used when precise orientationof tissue samples is required, such as with structures with lumen likethe intestine.

“Matrix material” as used herein refers to material used as a receptacleor foundation for the processing of histological samples. In oneembodiment, the matrix material provides structural integrity for theconstruction of the array. In another embodiment, the matrix materialassists in the processing of tissues for histological preparation. Thetissue matrices used in this disclosure are selected on the basis ofconsisting of a suitable blend of proteins, lipids, carbohydrates andwater to produce a matrix with low cross-reactivity to the samplescontained therein, and minimal shrinking and good cutting properties. Ina preferred embodiment of the invention, the matrix material isprocessed animal tissue.

“Processed animal tissue matrix” as used herein and throughout thisdisclosure refers to one type of tissue matrix, wherein the tissue iscomprised of animal tissue. One embodiment of this disclosure is aprocessed animal tissue matrix that can be used to create a biopsyarray. The processed animal tissue used in the processed animal tissuematrix comprise a suitable blend of proteins, fat, carbohydrates andwater to produce a matrix with minimal shrinking and good cuttingproperties. The processed animal tissue used is preferably selected onthe basis that it does not shrink or collapse during dehydration, andshould preferably not be so hard as to make sectioning difficult. In oneembodiment, the processed animal tissue matrix used performs well in allhistological preparative stages. In one embodiment of the disclosure,SPAM® is used to create the processed animal tissue matrix of thedisclosure. SPAM® processed pork exhibits minimal shrinking and goodcutting properties when used as a matrix material for biopsy arrays.

In one embodiment of the disclosure, a biopsy sample is obtained andfixed in a suitable fixative. The processed animal tissue matrix ispreferably fixed in a similar way. The sample is arranged in theprocessed animal tissue matrix to create the array. The array is capped,dehydrated, passed through a transitional solvent and embedded in abinding agent.

Embodiments of this disclosure have many advantages, including greatlyenhanced efficiency and speed for tissue testing; greatly decreased costfor multiple tissue testing; great economies in the use of tissuesamples, reagents and testing materials; great flexibility and ease ofconstructing biopsy arrays; lack of need for deparaffinization andre-embedding; ability to use tissues or cells as sources of samples forthe array; and the ability to use fresh, fixed or paraffinized biopsysamples. Employing a matrix during processing results in superiorconsistency in quality. Since all tissue biopsies can be treatedsimultaneously, one can reasonably expect less variation among themwithin the same paraffin block. In some embodiments, the disclosure alsoenables one to obtain serial sections (ribbons), for instance, as thinas 2 μm. In another embodiment, a biopsy array can be produced in lessthan 8 hours, more preferably between 8 and 24 hours. When using bankedtissue (transitional solvent), an arrayed block can be generated within3 hours for paraffin embedding, and in 10 to 15 minutes for frozenpreparations.

The matrix method allows parallel processing and sectioning of smalltissue samples of cylindrical shape of very small diameter by layingthem horizontally by the surgeon performing the biopsies within apre-sterilized matrix (chemically, by gamma irradiation, etc.). This isimportant when dealing with biopsies (e.g., prostate, thyroid, breast)when one has to report absolute and relative numbers (length/volume,percentage of malignant tissue).

In using a matrix according to the matrix method, during collection, thecore biopsies are placed directly into the preformed matrix and thenprocessed in any tissue processor or manually. Although the preformedmatrix is designed for manual processing, it is compatible with allexisting laboratory methods and automatic dehydration and infiltrationdevices. However, the use of microwave accelerated schedules of tissueprocessing is not recommended without prior testing. The matrix materialis able to withstand exposure to all the fixatives and volatile reagentstypically used in histology and will dehydrate and infiltrate in perfect“harmony” with the biopsies within it, without any distortions. Friablebiopsies are protected during processing, eliminating any loss ofprecious diagnostic material while serial sectioning and spreading aregreatly facilitated.

When dealing with prostate biopsies, the matrix is used in conjunctionwith rigid embedding cassettes. The matrix is aligned carefully in theparaffin block and the paraffin block is aligned carefully in the planeof sectioning. A corollary of employing a matrix during harvesting thebiopsies is that even minute fragments of tissue are not lost duringtransportation and the potential artifacts associated with processingare completely eliminated. Since all tissue biopsies are treatedsimultaneously (i.e., within the same paraffin block), one canreasonably expect less variation among them.

The matrix method makes the whole process very efficient and robust andmakes biopsy sectioning accessible to any average technician. The matrixensures that the biopsies are perfectly aligned, thus allowing veryprecise and expeditious diagnosis. The matrix method decreases by oneorder of magnitude the material, time and manpower expenses for prostatecore biopsy processing and analysis. However, the procedure does notimpose a supplementary burden on either the clinician harvesting thetissue or the laboratory staff, including the pathologist reading andinterpreting the slides, and is suitable for automated histologyanalysis. The burden of the pathologist is, therefore, significantlylessened (much smaller number of slides and much easier to read andquantify). The matrix method also enables-three dimensionalrepresentations of the neoplastic growth.

In another embodiment, the disclosure provides a tissue and or cellarray comprising a processed animal tissue matrix. The construction ofthe array is flexible and allows custom biopsy arrays to be built. Inone embodiment, the array is a multi-specimen array. “Multi-specimenarray” includes arrays composed of different samples from a singlespecies, the same cell or tissue type from different species, or thesame cell or tissue type from the same species at differentdevelopmental times. In a further embodiment, the array is a multi-usearray. “Multi-use array” includes arrays used to simultaneously screen anumber of biopsy samples, to create tissue-banking systems and tomanufacture sections and slides. One value of this technology is thattesting can be done on many samples in a timely manner with consistencyto allow for high-throughput analysis.

An additional embodiment of the disclosure provides a method forpreparing the biopsy arrays comprising a processed animal tissue matrix.At least one cell or tissue sample is manipulated and positioned in arecipient receptacle in the processed animal tissue matrix. It ispreferable that the receptacle in the processed animal tissue matrix isnot a complete puncture through the processed animal tissue, but a thinlayer of tissue remains at the bottom to create the receptacle. Afterthe samples are arranged in the matrix, processed animal tissue matrix,preferably in the similar preparative stage as the processed animaltissue matrix, is applied to the top of the matrix and acts like a lid.In one embodiment, the samples are arranged in the matrix at any pointin the histological preparation process. The sample-containing matrixcan then be processed using known histological techniques.

Method for Preparing a Multi-Specimen Biopsy Array within a Matrix

The method allows for the preparation of a “multi-specimen” biopsyarray. The method allows parallel processing of multiple biopsy samplesof cylindrical shape and very small diameter (0.5 to 1 mm) as typicallyobtained during core biopsy procedures. The method disclosed herein usesa matrix in which parallel grooves were pre-made. The biopsy samples arethen laid horizontally in the parallel grooves of the matrix.

The method is particularly advantageous when dealing with biopsies(e.g., prostatic, breast, thyroid) where one has to analyze multiplebiopsy samples and report the percentage of malignant tissue. Thepresent method makes the whole process quite easy and it is believedthat biopsy sectioning is now accessible to any average technician. Theburden of the pathologist is significantly lessened (much smaller numberof slides and much easier to read and quantify). The method alsosimplifies the tracking of the biopsy samples, which facilitates 3-Dreconstruction.

The present method is also useful for instances when a small number ofbiopsies (or only one) are taken. This is because by encasing the biopsywithin a matrix up front, the risk of losing it in the processor,fragmentation (sometimes the tissue harvested is already brittle orfriable: scars, hemorrhage, foci of necrosis, etc.) or distortion duringprocessing is completely eliminated.

FIG. 1A is a perspective view of a matrix for receiving biopsy samplesaccording to an embodiment of the disclosure. As shown in FIG. 1A, amatrix 100 includes a block 102 of matrix material 104. In the uppersurface 106 of block 102 a plurality of parallel receptacles in the formof grooves 120 a, 120 b, 120 c, 120 d, 120 e, and 120 f, have been made.The grooves 120 a-120 f are made by machining or cutting into the uppersurface 106 of block 102. The machining/cutting is relatively simple andrequires less than 5 minutes per matrix for preparing 12 grooves. Themachining of the matrix can be automated or performed manually.

The matrix can be prepared from a range of different materials. Thematerial used in preferably hydrophilic and exhibits minimal distortionsduring dehydration, clarification with solvents and paraffininfiltration. In particular, the material preferably has similarchemical properties with the biopsy samples to be placed therein. Someplant and animal tissues can be used as the matrix material. Forexample, plant material (e.g., sweet potato) and animal tissues (e.g.,beef liver) can be utilized to create the block. Synthetic materialshaving the desired properties can also be used. In preferredembodiments, the material chosen is widely available and inexpensive.

In an embodiment of the disclosure, a method is provided for preparing asuitable processed animal tissue matrix for use to embed a biopsy, suchas to create a biopsy array. The processed animal tissue matrix can beprepared using histological practices and procedures known to personsskilled in the art. In one embodiment, receptacles are formed in theprocessed animal tissue to create the matrix. It is preferable if thereceptacles are sized to accommodate the biopsy samples tightly. Thisaids in maintaining desired orientation of the sample within the matrix.

In a preferred embodiment, an off-the-shelf processed animal tissueproduct is utilized (e.g., SPAM®). This off-the-shelf processed animaltissue product material exhibited very good chemical and mechanicalproperties (it can be fixed, dehydrated, clarified, embedded andsectioned with ease). The matrix can be pre-stained for good opticalcontrast. Alternatively, the matrix is left unstained and the biopsiesare pre-stained during fixation.

The block of matrix material is optionally treated before machining. Forexample, the block of material can be fixed, dehydrated and infiltratedwith paraffin before machining them to the desired shape. Aftermachining, the matrix can be “reverse processed” to water. That is, asolvent is applied for extracting the paraffin, ethanol is then appliedfor extracting the solvent, then the matrix is rehydrated with water anda fixative is applied.

In one embodiment, the following method is used to prepare the processedanimal tissue material. The processed animal tissue is sectioned inslices, preferably no thicker than 10 mm to ensure good penetration withthe fixative. The processed animal tissue matrix is immersed in afixative. One fixative that can be used is fresh-buffered formalin (4%formaldehyde in 150 mM PBS-phosphate-buffered saline). It is preferableif the processed animal tissue is fixed in formalin for at least 24hours. After the processed animal tissue material is collected andfixed, the next step is to dehydrate the processed animal tissuematerial. In order to dehydrate the processed animal tissue material,progressive concentrations of ethanol in water are used. The last bathis in absolute ethanol. The processed animal tissue material ispreferably washed a minimum of three times with a transitional solvent.The processed animal tissue is then paraffin-embedded. A block of thematerial is then used for preparing a matrix. Receptacles are formedinto the processed animal tissue by drilling, machining, or punching.The matrices are then reverse-processed to water/fixative by removingthe paraffin with a transitional solvent, removing the solvent withethanol, removing the ethanol with water and stabilizing the animaltissue with a fixative. Techniques for reverse-processing are known topersons skilled in the art. The matrix is ready to receive the biopsysamples to create the array.

Alternatively, machining can be performed without prior paraffininfiltration. In this method, the block of matrix material is fixed,cryo-protected with sucrose, and then frozen. Techniques forcryo-protection, freezing and sectioning of frozen histologicalpreparations are known to persons skilled in the art. The frozen blockis then machined to the desired shape of the matrix. After machining,the matrix is then thawed in fixative.

In alternative embodiments, the matrix material is molded in the desiredshape and no machining is required. For example, where the matrixmaterial is processed animal tissue, a paste of the processed animaltissue can be poured into an appropriately shaped mold. After molding,the shape of the matrix can be stabilized using heat and/or chemicalfixation. The molding process thus eliminates the need for machining andsimplifies manufacturing of the matrix.

After preparation, a matrix can either be used immediately, or stored inan appropriate fixative until required. The use of fixative forlong-term storage is known to persons skilled in the art. When biopsysamples are received, a matrix is prepared or removed from storage. FIG.1B illustrates loading of biopsy samples into the matrix block of FIG.1A. As shown in FIG. 1B, biopsy samples 130 a, 130 b, 130 c, 130 d, 130e, and 130 f are placed one by one in grooves 120 a-120 f of matrix 100(see FIG. 1A) to form an array 101 of biopsy samples supported by matrix100.

An embodiment of this disclosure is a method of obtaining and treating abiopsy sample for use in the matrix. The cell or tissue sample can beprepared using histological practices and procedures known to personsskilled in the art. For instance, the samples can be fresh, fixed orembedded in a binding agent. It is preferred that the processed animaltissue matrix and tissue and/or cell samples are in a similarpreparative stage at the time the samples are arranged in the matrix,because some of the chemicals used during processing are incompatible.For example, if the biopsy sample is fresh, then the matrix shouldpreferably also be fresh. If the biopsy sample is fixed in ethanol, thenthe matrix should preferably also be in ethanol. If the biopsy sample isstored in a transitional solvent, then the matrix should preferably bein a transitional solvent. If the biopsy sample is paraffin embedded,then the matrix is preferably embedded in paraffin. Alternatively, asnoted below, the processed animal tissue matrix can be in a subsequentpreparative stage to the tissue and/or cell sample.

As shown in FIG. 1B, the biopsy samples are preferably marked at one endwith, e.g., ink (132), to identify the orientation of the samplerelative to the entry point of the needle. The biopsy samples arepreferably loaded into the matrix in the same orientation. The matrixitself may also be marked or shaped such that the order of the biopsysamples can also be identified.

The biopsy samples are collected and then fixed. Suitable fixatives andprocedures for good quality fixation are familiar to those skilled inthe art. After the tissue is collected and fixed, the next step is todehydrate the tissue sample. In order to dehydrate the tissue sample,progressive concentrations of ethanol in water are used. The last bathis in absolute ethanol. The tissue sample is preferably washed a minimumof three times with a transitional solvent. The tissue samples can thenbe trimmed to fit in the matrix. If desired, it is possible to formreceptacles into the matrix and the samples can be fitted into theprocessed animal tissue matrix during any stage. This allows significantsavings in the labor involved when one has to process large numbers ofsamples.

In a preferred embodiment, as shown in FIG. 1B, grooves 120 a-120 f ofmatrix 100 are the same size and shape, and biopsy samples 130 a-130 fare also of the same size and shape. Thus, matrix 100 presents thebiopsy samples at the same depth. This allows a single section of thearray to intercept all of the biopsy samples (see, e.g., FIG. 1F). Thisreduces waste of the tissue and allows for significantly more tissue tobe left in the resulting block for archival purposes.

FIG. 1C illustrates application of a sponge to the matrix block of FIG.1A. After the biopsy samples are loaded, a sponge 140 is placed on topof the array 101 of biopsy samples supported by matrix 100 as shown inFIG. 1C. This prevents curling of the biopsy samples during processing.In alternative embodiments, a sheet of the same matrix material may beused in place of sponge 140.

In one embodiment of the disclosure, a biopsy sample is obtained andprepared. The processed animal tissue matrix is preferably in thesimilar or subsequent preparative stage as the samples. At least onesample is arranged in the processed animal tissue matrix to create thearray. The array is then preferably capped and embedded in a bindingagent.

FIG. 1D illustrates the matrix block of FIG. 1A and sponge of FIG. 1C incombination with a tissue cassette. Sponge 140 and the array 101 ofbiopsy samples supported by matrix 100 are then introduced in standardtissue processing cassette 150 as shown in FIG. 1D. The upper 152 andlower 154 sides of the standard tissue processing cassette 150 aretypically perforated to allow passage of the reagents through thecassette 150. Loading the biopsy samples into the matrix and loadinginto the standard tissue processing cassette can be achieved in lessthan about five minutes for up to about twelve samples. The loadedtissue cassette can then be processed using standard tissue processingequipment and techniques. Preferably, the matrix is placed in theapparatus with the grooves facing upward to reduce the chance ofmigration of the biopsy samples. During processing, the biopsy samplesand matrix are typically embedded in paraffin wax in preparation forsectioning. Processing and embedding of the loaded matrix are carefullyperformed in order to maintain the flatness of the matrix in order toallow for easy sectioning.

FIG. 1E is a schematic representation of a slide prepared from a sectionof the matrix block and biopsy samples of FIG. 1A according to anembodiment of the disclosure. After tissue processing and embedding, theembedded array 101 of biopsy samples supported by matrix 100 issectioned in the standard fashion using a microtome. Sections canreadily be cut between 2 μm and 10 μm in thickness. With the embeddedarray 101 of biopsy samples supported by matrix 100, sections can beobtained through the entire length of each biopsy sample in the samesection, allowing single slides to be created, which allow for full-coreanalysis of multiple samples. The sections are placed on slides usingconventional tissue processing techniques.

As shown in FIG. 1E, a slide 160 includes a section 162 under a coverslip 164. The section 160 includes full-length sections through eachbiopsy sample 130 a, 130 b, 130 c, 130 d, 130 e, and 130 f separatedfrom one another by matrix material 104. The biopsy samples can bereadily identified and distinguished from the interspersed matrixmaterial 104. The slide allows for analysis of each of the biopsysamples 130 a, 130 b, 130 c, 130 d, 130 e, and 130 f along the fulllength of each core. FIG. 1F is a picture of a sample slide preparedutilizing the matrix block of this disclosure. The slides can be usedfor histological procedures, including quantitative analyses andparallel processing.

Alternative Matrix Configurations

The matrices of this disclosure can be readily manufactured in a rangeof shapes suitable for supporting biopsy samples. Alternative matrixdesigns can provide features and advantages for supporting biopsysamples. A range of alternative matrix designs is illustrated in FIGS.2A-2D.

FIG. 2A is a perspective view of an alternative matrix block accordingto an embodiment of the disclosure. As shown in FIG. 2A, a matrix 200 aincludes a block 202 a of matrix material 204 a. In the upper surface206 a of block 202 a, a plurality of parallel receptacles in the form ofgrooves 220 a, have been made. The plurality of grooves 220 a is made bymolding or machining/cutting into the upper surface 206 a of block 202a. In the embodiment shown in FIG. 2A, the grooves 220 a are rectangularin section and closed ended. Grooves 220 a are the same size and shape.Alternatively, the grooves can be made at various depths and widths, inorder to accommodate the various types of biopsies.

FIG. 2B is a perspective view of an alternative matrix block accordingto an embodiment of the disclosure. As shown in FIG. 2B, a matrix 200 bincludes a block 202 b of matrix material 204 b. In the upper surface206 b of block 202 b, a plurality of parallel receptacles in the form ofgrooves 220 b, have been made. The plurality of grooves 220 b ispre-made by molding or machining/cutting into the upper surface 206 b ofblock 202 b. In the embodiment shown in FIG. 2B, the grooves 220 b arerectangular in section and open ended. The grooves 220 a have differentsizes to accommodate different sizes of biopsy samples while presentingthem at the same height for sectioning.

Grooves can be prepared having a variety of different sectional shapes.For example, in an alternative variation, the grooves can have a“triangular” shape, in order to accept any size of biopsy sample. FIG.2C is a perspective view of an alternative matrix block according to anembodiment of the disclosure. As shown in FIG. 2C, a matrix 200 cincludes a block 202 c of matrix material 204 c. In the upper surface206 c of block 202 c, a plurality of parallel receptacles in the form ofgrooves 220 c, have been made. The plurality of grooves 220 c ispre-made by molding or machining/cutting into the upper surface 206 c ofblock 202 c. In the embodiment shown in FIG. 2C, the grooves 220 c aretriangular in section and open ended. Grooves 220 c are the same sizeand shape. Alternatively, the grooves can be made at various depths andwidths.

FIG. 2D is a perspective view of an alternative matrix according to anembodiment of the disclosure. As shown in FIG. 2D, a matrix 200 dincludes a block 202 d of matrix material 204 d. In the block 202 d, aplurality of parallel receptacles in the form of bores 220 d, have beenmade. The plurality of bores 220 d are pre-made by molding ormachining/cutting into the block 202 d. In the embodiment shown in FIG.2D, the bores 220 d are circular in section and open at both ends. Bores220 d are the same size and shape. Alternatively, the bores can be madeat various depths and widths, in order to accommodate the various typesof biopsies.

In order to insert biopsy samples into the matrix of FIG. 2D, the matrix200 d is placed in a rig that allows little scoops (half cylinder inshape) to be introduced through the bores 220 d and protrude at leastthe length of the expected biopsy samples. The samples are placed in thegroove of the scoop. Another half-cylinder scoop is placed as a “lid”over the biopsy and both scoops are introduced within the matrix 200 d.After the correct location is reached, both scoops are extracted,leaving the biopsy samples inside the bores 220 d of the matrix 200 d.The matrix with the enclosed biopsies is then introduced into a tissuecassette and processed as usual. This method can be used on theoperating table where the biopsies are taken. This would eliminate“chain of custody” issues. In an alternative embodiment, a “modified”biopsy needle is used to introduce the biopsy sample directly into thematrix. The biopsy needle may be single use or multiple uses. However,if the biopsy needle is to be used after introduction into the matrix,the matrix is required to be sterile and the matrix material must bebiocompatible to avoid contaminating the needle.

In alternative embodiments, individual biopsy samples are mounted withinindividual matrices of identical external shape and size. The matricescan have different size grooves/bores for receiving the biopsy samplesso long as the samples are presented in the same plane. The individualmatrices can be processed separately and then grouped in variouscombinations during embedding. The identical size of the matrices wouldallow the resulting composite block to have all biopsies located in thesame plane of sectioning.

Alternative Matrix Configuration and Loading Apparatus

One advantage of the matrix material of this disclosure is that thematerial is relatively flexible. The matrix material has a certaindegree of flexibility when hydrated. The flexibility of the materialallows the design of matrix configurations that facilitate the loadingand securing of biopsy samples within the matrix. As shown in FIGS.3A-3C, a matrix can be prepared having channels provided with a smallopening/slot to the upper surface. To load biopsy samples, the matrix isplaced over a curved surface. This causes the openings to be enlarged tofacilitate the introduction of the biopsy sample. For example, thebiopsy needle can be used to introduce the sample directly to thematrix. After introduction of the sample, the matrix is straightened,such that the opening diminishes in size, securing the sample in place.When the needle is extracted, it will leave the biopsy behind.

FIG. 3A is a perspective view of an alternative matrix block accordingto an embodiment of the disclosure. As shown in FIG. 3A, a matrix 300includes a block 302 of matrix material 304. In the upper surface 306 ofblock 302, a plurality of parallel receptacles in the form of channels320 a, 320 b, 320 c, 320 d, 320 e, 320 f, 320 g, 320 h, and 320 i havebeen made. The channels 320 a-320 i are made by cutting, machining ormolding the upper surface 306 of block 302. As shown, the channels 320a-320 i have a narrow opening/slot through the upper surface 306. Thelower surface 308 is provided with a plurality of features 322 in theform of dovetail slots.

FIG. 3B is a schematic view of a method and apparatus for loading biopsysamples into the matrix block of FIG. 3A according to an embodiment ofthe disclosure. The matrix material has a degree of flexibility whenhydrated. As shown in FIG. 3B, features 322 are engaged with a roller350. Roller 350 forces matrix 300 into a curved shape. The curved shapeof matrix 300 increases the size of the openings of channels 320 a-320i. A plurality of biopsy samples 330 is introduced into the plurality ofchannels 320 a-320 i while the openings of the channels 320 areenlarged. As the channels 320 a-320 i are sequentially loaded, matrix300 moves over roller 350 onto platform 352. Matrix 300 straightens outon platform 352, causing the openings of channels 320 a-320 i to becomesmaller, thereby trapping biopsy samples 330 within channels 320 a-320i. Optionally, an adhesive/sticky material 324 can also be provided inchannels 320 a-320 i to further secure biopsy samples 330. One exampleof adhesive/sticky material that can be used for securing the biopsiesin place is a dilute solution of gum Arabic. Other adhesive materialsare known to those skilled in the art.

After loading of the biopsy samples 330, the matrix 300 and biopsysamples 330 are processed as previously described. FIG. 3C illustratesthe matrix block of FIG. 3A in combination with a tissue cassette andsponge. A sponge 340 is placed on top of matrix 300, and the matrix 300,sponge 340 and biopsy samples 330 are then introduced in standardtissue-processing cassette 360 as shown in FIG. 3D. The upper 362 andlower 364 sides of the standard tissue-processing cassette 360 aretypically perforated to allow passage of the reagents through thecassette 360.

The loaded tissue cassette can then be processed using standardtissue-processing equipment and techniques. Preferably the matrix isplaced in the apparatus with the grooves facing upward to reduce thechance of migration of the biopsy samples. During processing, the biopsysamples and matrix are typically embedded in paraffin wax in preparationfor sectioning. Processing and embedding of the loaded matrix arecarefully performed in order to maintain the flatness of the matrix inorder to allow for easy sectioning.

FIG. 3D is a perspective view of a variation 300 d of matrix block 300of FIG. 3A, according to an embodiment of the disclosure. As shown inFIG. 3D, a matrix 300 d includes a block 302 of matrix material 304. Inthe upper surface 306 of block 302 a plurality of parallel receptaclesin the form of channels 320 a, 320 b, 320 c, 320 d, 320 e, 320 f, 320 g,320 h, and 320 i have been made. The channels 320 a-320 i are made bycutting, machining or molding the upper surface 306 of block 302. Asshown, the channels 320 a-320 i have a narrow opening/slot through theupper surface 306. The lower surface 308 is not, in this case, providedwith dovetail slots.

Alternative Tissue Matrix Material

In an alternative embodiment, a versatile synthetic tissue matrix forhandling, processing and sectioning multiple biopsies or explants issynthesized from various ingredients as specified below. The compositionof the synthetic tissue matrix is essentially a “man-made meat/tissue.”The synthetic tissue matrix is made by combining: 2% to 14% animalprotein (preferably myofibrillar, e.g., pork, chicken, or fish surimi);0.1% to 5% Animal fat; 2% to 15% Vegetable oil (e.g., palm, corn,sunflower, olive); 2% to 15% Glycerin; 1% to 10% antifoaming agent(e.g., agarose, etc.); and inorganic buffers: e.g., phosphate: 10-600mM. In a preferred embodiment, the synthetic tissue matrix is made bycombining: 2% animal protein (preferably myofibrillar, e.g., surimi); 5%Animal fat; 5% Vegetable oil (e.g., palm, corn, sunflower, olive); 5%Glycerin; 5% antifoaming agent (e.g., agarose); and 200 mM inorganicphosphate buffer.

The mixture is thermally gelled by heating at 60° C. to 95° C., morepreferably 65° C. to 80° C., or enzymatically by using transglutaminase.These methods for gelling animal proteins are known to persons skilledin the art. Alternatively, complex carbohydrates are included forgelling (e.g., alginate, carrageenan, konjac gum, etc.), which can beionically gelled (Ba, Zn, Ca, Sr, etc.—as salts: CaCl2, BaCl2, ZnSO4,etc.) by submerging the mixture in a water-based solution containing thesalts in a concentration of 0.2% to 15%, preferably 2% to 5%.Alternatively, by incorporating in the mixture one or more of thevarious divalent cations (typically Ca), they are slowly released andthe gel is formed within minutes of mixing. Other methods known in theart for creating gels can also be used for gelling the synthetic tissuematrix disclosed above. The ratios of the various components (proteins,lipids, carbohydrates) of the gelling mixture can be adjusted formimicking the type of tissue to be sectioned. It is preferred that thesynthetic tissue matrix be as hard, or slightly harder, than the tissueof interest. When used for analysis of biopsy samples, the material canbe referred to as HISTOSPAM matrix material.

The protein gel can be maintained in viscous form for extended periodsof time and gelled when needed. In an alternative embodiment, when rapiddiagnosis is desired, the biopsy or explants (e.g., skin, gastric,breast) can be positioned and included in a mass of the viscous proteingel with a composition suitable for the tissue of interest, oriented asneeded and the whole assembly (biopsy and surrounding protein gel) ispolymerized by spraying/immersion with a crystalloid solution aspreviously described (Ba, Zn, Ca, Sr, etc.—as salts: CaCl2, BaCl2,ZnSO4, etc., in a water-based solution containing the salts in aconcentration of 0.2% to 15%, preferably 2% to 5%). The “solidified”assemblies (biopsy plus gelled proteic mixture) can be furthersnap-frozen for cryosectioning and histodiagnostics. The techniques forsnap-freezing, cryosectioning and histodiagnostics are well known topersons skilled in the art. Alternatively, the “solidified” assemblies(biopsy plus gelled proteic mixture) can be further chemically fixed,dehydrated, infiltrated in paraffin, sectioned and used forhistodiagnostics. The techniques for chemical fixation, dehydration,paraffin infiltration, sectioning and histodiagnostics are well known topersons skilled in the art. In yet another embodiment, the protein gelcan be molded, extruded or injected for creating permanent (gelled)matrices. The matrices can be frozen or chemically fixed for later use.

They can also be chemically sterilized or gamma-irradiated for long-termstorage. Additionally, pre-made matrices already embedded in paraffincan be machined and shaped as recipient blocks for acceptingparaffin-embedded tissue fragments extracted from donor blocks.Typically, the gelled matrix is formed or shaped such that it providespre-made receptacles for receiving the biopsies or explants (e.g., skin,gastric, breast) as disclosed above. The shape of the pre-formedreceptacles within the matrix is selected based on the shape of thetissue sample to be processed: rounded or rectangular grooves forcylindrical shapes; tru-cut biopsies (prostate, breast, thyroid, gauge20 and larger), or for fine-needle aspirates; or free-form for irregulartissue samples. The biopsies or tissue fragments can be laidhorizontally (biopsy chip) or vertically. Another shape of the pre-madematrix is similar with a brush-shape or funnel-shape suitable forirregular-sized tissue fragments (tumor margins, shavings in Mohssurgery, etc.), thereby allowing the irregular fragments to be easilypositioned in the appropriate angle. Once positioned in the premadereceptacles, the biopsies or explant samples can be sectioned in acryostat (for “real-time” diagnostic: breast, skin) or fixed andprocessed through paraffin as described above.

One other application benefiting from the multiplex sectionable matrixis for the stereotaxic mapping of large tissue/tumors/organs removedduring surgery. The rule is to embed and section the fragment either intoto, or only partially, but using a systematic approach in selectingthe areas to be examined (to decrease the workload but keeping at aminimum the risk of missing cancer).

First Example

The aim of the study was to assess the suitability and effectiveness ofour new method of prostate biopsy collection, processing and analysisusing the grooved matrix block as disclosed herein within a pilot trial,as well as to start a comprehensive tissue archive for furthermulti-center prospective longitudinal cohort studies.

Method: A multiplex grooved matrix block 300 d as disclosed herein wasconstructed from a protein gel (see FIG. 3D) and used for aligning thespecimens by the urologist who collected the biopsies from 30 patientssuspected of prostate cancer. Inclusion criteria for participants in thetrial were based on clinical (positive DRE) or biochemical (PSA>2.5ng/ml) suspicion of prostate cancer. Up to 12 biopsy cores per patient(gauge 18) were collected with an ultrasound-guided biopsy gun in asingle matrix and placed in a matrix 300 d as shown in FIGS. 4A-4C. Asshown in FIG. 4A, the biopsy core 400 was exposed at the tip of theneedle 410 of the biopsy gun 412. As shown in FIG. 4B, the biopsy core400 was deposited in a groove (320 a-320 i) of the matrix 300 d by agentle in-axis rotation of the needle 410. The loaded matrix was labeledand placed between two foam biopsy pads in a standard histology cassette(see, e.g., FIG. 3C), immersed in neutral-buffered formalin, and sent tothe pathology department. The individual cores were placed in matrix 300d at specified positions and in the same orientation with respect to theentry point of the biopsy needle into the prostate. The location of eachcore 400 was marked with a number on a prostate diagram map for eachbiopsy protocol sheet, allowing unambiguous and easy location of thetumor and estimation of its size and extension.

Processing of biopsy-loaded matrices was performed in a standardizedfashion, and consisted in: fixation for 24±2 hours with 4% formaldehyde,dehydration in graded ethanols, clearing through transitional solvents,infiltration and embedding in paraffin. Semi-serial sections at 4-5 μmwere mounted on positively charged glass slides. Every fifth section wasstained with hematoxylin-eosin (HE). When needed, immunohistochemistry(IHC) was performed with PIN cocktail containing mouse anti-p63 andrabbit anti-P504S (Alpha-Methylacyl-CoA Racemase or AMACR) primaryantibodies at dilution 1:100 (CO001K-05—Zytomed Systems, Berlin,Germany) and visualized with Envision Flex™/HRP and DAB+(DAKO, Glostrup,Denmark).

The main outcome measures were the aggregated lengths of biopsy corefragments on slides at different levels and of the remaining tissue inthe paraffin blocks; the time and material expenses for histopathologyand immunohistochemistry using our new approach compared to theclassical procedures.

Results and limitations: The results of the study are illustrated byFIGS. 4C-4E. FIG. 4C, shows a plurality of biopsy cores 400 loaded in amatrix block 300 d. The loaded matrix block 300 d is shown in processingcassette 420. FIG. 4D shows a grid array of sections 402 of biopsy cores400 in a paraffin block 430. For each patient, 22 semi-serial sectionswere cut and the remaining material was left in the paraffin block 430).The sectioning time for each paraffin block (i.e., patient), regardlessof the number of biopsy cores within, did not exceed 15 minutes. FIG. 4Eshows a grid array of sections 402 of biopsy cores 400 on a slide 440prepared for histological analysis. Note that, as shown in FIG. 4E, morethan 100 sections are arrayed on a single microscope slide at a densityof 20 or more sections per cm².

The biopsies did not curl and maintained their correct position, evenwhen they were fragmented at the time of placement into the matrix. Theaverage length (±SD) of processed biopsy cores was 13±4 mm (n=299). Theaverage aggregate length of biopsy fragments per patient (±SD) was:131.9±25.3 mm for 10 cores (n=27), 130.0±9.1 mm for 12 cores (n=2), and99 mm for 6 cores (n=1). The biopsies did not show curling duringprocessing, remained properly oriented, and maintained intact tissuerelationships even when the cores were fragmented. The aggregate corelength was approximately 80% of the fresh core length, however, thislikely represents normal core shrinkage during processing; hence,substantially all of the core was represented in the sectioned sample.Sectioning was greatly facilitated by the matrix employed and at least50% of the bioptic material was saved in the paraffin block. Reportingof the histopathological findings was made in a quantitative fashion,and spatial representations of the neoplastic tissue were recorded.

From the 30 patients included in this study, three presented benignprostate hyperplasia (BPH), four atypical small acinar proliferation(ASAP), two intraepithelial neoplasia (PIN), one ASAP and low-grade PIN,seventeen prostate adenocarcinoma, one nonspecific nodular chronicinflammatory disease, and two were found free of prostate disease. Theaverage length of cancer regions per patient (±SD) was 26.0±26.8 mm. Acomplete biopsy core was reconstructed from multiple microscope fieldsat ×10 magnification from two sections 5 μm apart stained with HE andPIN cocktail, respectively. Other sections from the same patientpresented atypical cribriform glandular patterns suggestive of highgrade PIN at three magnifications (×10, ×20, ×40) (FIG. 2, lower row),with ductal epithelial cells positive for AMACR (cytoplasmic) andnegative for p63 (nuclear).

The study found a statistically significant positive correlation betweenPSA levels and the numbers of positive cores for each patient (r=0.580,p=0.00097, n=29), a correlation between PSA and Gleason scores (r=0.409,p=0.116, n=16), and a negative correlation between prostate volumes, asestimated via TRUS, and numbers of positive cores (r=−0.256, p=0.181,n=29), although the latter two were not statistically significant. Therewere statistically significant associations between positive/negativeDRE and TRUS or presence/absence of prostate cancer (p=0.000203 andp=0.00298, respectively, one-tailed Fisher exact test), while TRUS wasnot significantly associated with prostate cancer (p=0.5535, one-tailedFisher exact test). The volume of cancer, computed from the percentagesof positive areas on biopsy cores, was correlated with PSA levels(r=0.825, p<0.0001, n=16), number of positive cores (r=0.567, p=0.022,n=16) and Gleason scores (r=0.484, p=0.05741, n=16), while the totalprostate volume was not.

Conclusions: The tissue matrix employed in arraying the tissue biopsiesperformed flawlessly. This matrix method conferred superior speed andreliability, while reducing laboratory expenses significantly. Readingthe slides was regarded by the pathologists as straightforward andaccurate, particularly regarding annotations and rcvicw of suspiciousareas. This technique does not require any changes in the processingschedules; paraffin blocks can be obtained without any difficulty andserial sections (ribbons) as thin as 3 μm can be obtained fairly easily.As customary, when dealing with prostate biopsies, one has to aligncarefully the plane of sectioning. A corollary of employing a matrixduring harvesting the biopsies is that even minute fragments of tissueare not lost during transportation and the potential artifactsassociated with processing are completely eliminated. Since all tissuebiopsies are treated simultaneously (i.e., within the same paraffinblock), one can reasonably expect less variation among them.

The multiplex method of harvesting, processing and reporting of prostatebiopsies using the tissue matrix disclosed herein is an easilyapplicable, cost-effective method, provides tumor location informationand creates consistent duplicate arrays for analysis and researchpurposes. Unlike other methods, it can be used efficiently for parallelquantitative analysis of various biopsy samples.

Comparative Example

A study was conducted to evaluate multiplex processing and analysisusing a matrix block as disclosed herein in comparison to conventionaltechniques. Thirty-six patients suspected of prostate cancer wererandomly assigned in two groups (conventional and matrix block) and upto 24 biopsy cores per patient (gauge 18) were collected with anultrasound-guided biopsy gun. The biopsies were collected in separatevials and processed individually for the first 17 patients while thefollowing 19 were collected and processed in groups of six biopsies byusing the matrix block. All histopathology processing, sectioning andstaining maneuvers, the yield of bioptic material examined, as well asreading the slides, were timed and recorded.

Results: The duration of harvesting the biopsies was similar using bothmethods (8.3 vs. 7.64 minutes). Significant savings were recorded interms of total processing time (105.0 minutes per patient forconventional vs. 20.03 for matrix block) while the duration of readingthe slides was unaffected (21.6 vs. 22 minutes). Unlike conventionalmethods, the matrix block maintained intact tissue relationships evenwhen the cores were fragmented. On average, the length of the coresobtained (and examined) on slides was 66.4% of the initial biopsy(8.6/13.0 mm) when using the matrix block vs. 52.5% when conventionalmethods were employed (6.4/12.2 mm). Reporting of the histopathologicalfindings was made in a quantitative fashion, and spatial representationsof the neoplastic tissue were recorded.

Conclusions: The multiplex method of harvesting, processing andreporting of prostate biopsies using a matrix block as disclosed hereinis an easily applicable, cost-effective method, provides tumor locationinformation and creates consistent duplicate arrays for analysis andresearch purposes. Unlike other methods, it can be used efficiently forparallel quantitative analysis of various biopsy samples.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

The foregoing description of preferred embodiments of this disclosurehas been provided for the purposes of illustration and description. Itis not intended to be exhaustive or to limit the disclosure to theprecise forms disclosed. Many embodiments were chosen and described inorder to best explain the principles of the disclosure and its practicalapplication, thereby enabling others skilled in the art to understandthe disclosure for various embodiments and with various modificationsthat are suited to the particular use contemplated. It is intended thatthe scope of the disclosure be defined by the claims and theirequivalents.

The invention claimed is:
 1. A matrix suitable for receiving a tissuesample prior to or after paraffin embedding, said matrix comprising: ablend of 2-14% w/v animal protein, 2.1-20% w/v one or more lipidsselected from the and group consisting of animal fat and vegetable oil,2-15% w/v glycerin, water, a gelling agent, wherein the gelling agent isionically gelled, and an inorganic buffer in a concentration in therange of from 10 to 600 mM; and preformed receptacles in the matrixconfigured to receive at least one tissue sample.
 2. The matrixaccording to claim 1, wherein the receptacles are horizontally arrangedparallel grooves.
 3. The matrix according to claim 1, in combinationwith a tissue cassette and sponge.
 4. The matrix according to claim 1,wherein the matrix is configured to receive fresh tissue samples in thepreformed receptacles.
 5. The matrix of claim 1, wherein the matrixfurther comprises an antifoaming agent.
 6. The matrix of claim 5,wherein the blend comprises 1-10% w/v of the antifoaming agent.